Ischemia/reperfusion injury (IRI) to donor organs substantially enhances the immunogenicity of grafts and increases the rate of acute and chronic allograft rejection. Grafts with more severe IRI also have significantly worse long-term survival. Thus, IRI remains the major obstacle in transplantation today, but there are currently no standard treatments for IRI post-transplantation. Therefore, studies which lead to the development of novel strategies to attenuate the effects of IRI are much needed in the current era of transplantation. Our overall hypothesis is that IRI in the graft activates allograft derived dendritic cells (ADDC) which become the critical link between the innate immunity and alloimmunity, ultimately enhancing the allogenicity of the graft. ADDC are the most potent antigen-presenting cells. Extensive preliminary data from our laboratory using various transgenic models have led to the novel observation that preventing this injury could diminish the immunogenicity of ischemic organs and promote engraftment. The overarching goals of this project are: 1) to investigate the mechanisms by which IRI enhances the immunogenicity of allografts by increasing the allogenicity of ADDC, and 2) to identify novel protective strategies to minimize IRI, with the ultimate goal of promoting long-term allograft acceptance. In AIM 1, we will examine the mechanisms by which activated ADDC by IRI link innate and alloimmunity. We will employ T reg and alloreactive transgenic mice to study whether and how the ischemia/anti-ischemic strategy affects the generation of T regs vs. alloreactive T cells. In AIM 2, we will explore the mechanisms by which ischemia-induced TLR2/TLR4 activation results in higher DC allostimulatory capacity and trafficking properties. We will identify the key signaling molecules in ischemic DC upstream of NFkB and downstream of TLR4 and TLR2, with particular focus on MyD88 and TRIF. Ischemic DC will be evaluated for their cytokine elaboration profiles, allostimulatory capacity, and chemokine dependent in vitro and in vivo trafficking patterns. The organ shortage has become a major obstacle hampering the success of organ transplantation worldwide, prompting clinicians to expand their criteria for the acceptance of marginal organs, which are more susceptible to IRI. Our proposed comprehensive studies will provide highly innovative data and insights into the pathophysiology of IRI-induced activation of innate and alloimmunity. This research will identify novel therapeutic targets and set forth novel strategies and innovations in the areas of donor management to attenuate the deleterious effects of IRI which are urgently needed in the current era of transplantation. We believe that this research, supported by the wealth of preliminary data, innovative approaches, sophisticated models and the commitment and expertise of the investigators, has all the necessary elements to achieve the stated goals. The findings from this proposal could be easily translated into clinical practice and have a significant impact on reducing the burden of IRI in clinical transplantation.